RESEARCH ARTICLE

Immunogenicity of Isogenic IgG in Aggregates and Immune Complexes J. Benjamin St. Clair1,2,3, Thiago Detanico1,3, Katja Aviszus1,3, Greg A. Kirchenbaum3¤a, Merry Christie4¤b, John F. Carpenter4, Lawrence J. Wysocki1¤c*

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OPEN ACCESS Citation: St. Clair JB, Detanico T, Aviszus K, Kirchenbaum GA, Christie M, Carpenter JF, et al. (2017) Immunogenicity of Isogenic IgG in Aggregates and Immune Complexes. PLoS ONE 12 (1): e0170556. doi:10.1371/journal.pone.0170556 Editor: Aftab A. Ansari, Emory University School of Medicine, UNITED STATES Received: May 13, 2016 Accepted: January 7, 2017 Published: January 23, 2017 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Funded by National Institutes of Health/ National Institute of Allergy and Infectious Disease: R01AI033613 (LJW) R21AI121980 (LJW), R03AI088408 (LJW) and National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases F30DK091102 (JBS). Competing Interests: The authors have declared that no competing interests exist.

1 Department of Biomedical Research, National Jewish Health, Denver CO, United States of America, 2 Medical Scientist Training Program, University of Colorado School of Medicine, Denver, Colorado, United States of America, 3 Integrated Department of Immunology, National Jewish Health and University of Colorado School of Medicine, Denver, Colorado, United States of America, 4 Department of Pharmaceutical Sciences, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado, United States of America ¤a Current address: Department of Infectious Disease, University of Georgia College of Veterinary Medicine, Athens, Georgia, United States of America ¤b Current address: Office of Biotechnology Products, Center for Drug Evaluation and Research, US Food and Drug Administration, Silver Spring, Maryland, United States of America ¤c Current address: Department of Immunology, University of Colorado School of Medicine, Denver, Colorado, United States of America * [email protected]

Abstract A paradox in monoclonal antibody (mAb) therapy is that despite the well-documented tolerogenic properties of deaggregated IgG, most therapeutic IgG mAb induce anti-mAb responses. To analyze CD4 T cell reactions against IgG in various physical states, we developed an adoptive transfer model using CD4+ T cells specific for a Vκ region-derived peptide in the hapten-specific IgG mAb 36–71. We found that heat-aggregated or immune complexes (IC) of mAb 36–71 elicited anti-idiotypic (anti-Id) antibodies, while the deaggregated form was tolerogenic. All 3 forms of mAb 36–71 induced proliferation of cognate CD4+ T cells, but the aggregated and immune complex forms drove more division cycles and induced T follicular helper cells (TFH) development more effectively than did the deaggregated form. These responses occurred despite no adjuvant and no or only trace levels of endotoxin in the preparations. Physical analyses revealed large differences in micron- and nanometer-sized particles between the aggregated and IC forms. These differences may be functionally relevant, as CD4+ T cell proliferation to aggregated, but not IC mAb 36–71, was nearly ablated upon peritoneal injection of B cell-depleting antibody. Our results imply that, in addition to denatured aggregates, immune complexes formed in vivo between therapeutic mAb and their intended targets can be immunogenic.

Introduction The widespread administration of therapeutic monoclonal antibodies (mAb) has revealed a paradox in the immune response to immunoglobulin-derived antigens. While the historical literature would suggest that soluble, bivalent IgG is profoundly tolerogenic and suppresses Ig-

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specific humoral responses, therapeutic mAbs can be immunogenic and commonly elicit antiId responses in some percentage of recipients, particularly in populations treated for autoimmune diseases. The clinical ramifications of anti-mAb have varied in individual clinical trials, but meta-analyses have confirmed decreased therapeutic efficacy and increased adverse events such as hypersensitivity reactions [1]. To prevent the development of high affinity antibodies directed against therapeutic mAb, researchers and physicians have adopted a number of strategies, with varying practical and theoretical costs and benefits, many of which target CD4+ T cell responses to Ig-derived peptides [1–4]. This focus on the immunogenicity, or tolerogenicity, of Ig for CD4+ T cells is based upon a historical dichotomy in the literature. Dresser first revealed the tolerogenicity of deaggregated, heterologous gamma globulin in 1961 [5–7]. Chiller, Habicht, and Weigle demonstrated that both mouse T helper cells and B cells could be tolerized by polyclonal, deaggregated human gamma globulin, and that T cell tolerance was both long lived and dominant when thymocytes were adoptively transferred into irradiated animals along with normal bone marrow [8–10]. In contrast, Janeway and Paul reported the augmentation of anti-idiotypic antibody production to a hapten-conjugated antibody if mice received a hapten-targeted antisera [11]. This suggested a potential adjuvant role for immune complexes, however the experiment was complicated by the hapten-conjugation to the targeted antibody which led to low anti-idiotypic production without antisera, a potential consequence of novel T-epitopes, aggregation, or endotoxin [12]. In a more recent study, Reitan and Hannestad found that a pentameric IgM form of a monoclonal Ig without adjuvant or endotoxin was immunogenic, while the IgG form was not, even after multiple injections [13–15]. Finally, inclusion of certain peptides into the structure of IgG renders them tolerogenic for CD4+ T cells and mitigates pathology in a mouse model of autoimmune disease [16–25]. Despite evidence for the tolerogenic properties of IgG, therapeutic IgG mAbs often elicit IgG antibody directed against the infused mAb [26–30]. This occurs even when the therapeutic mAb are encoded by entirely human Ig genes. While the CD4+ T cell repertoire attains selftolerance to germline Ig V region sequences, somatically generated diversity arising at boundaries of V region genes during B cell development or throughout the entire V region via somatic hypermutation is potentially antigenic [13–15, 31–40]. In cases where it is antigenic, this somatic diversity may provide an avenue of T cell help to any B cell specific for the idiotype of a therapeutic mAb. However, antigenic peptide sequences in Ig alone may be insufficient to elicit a productive anti-Id response, which has led researchers to hypothesize that mAbs are more likely to be immunogenic if they are aggregated during handling, targeted to a cell surface antigen, or engaged in immune complexes [41–49]. Prior studies in experimental models generally assessed CD4+ T cell reactions to IgG under circumstances in which the IgG could not form immune complexes in vivo, either because the IgG was polyclonal and nonspecific or because the cognate antigen for a monoclonal IgG was not present [38, 40, 50–65]. And in most of these studies, analyses were limited because they involved wildtype i.e. nontransgenic T cells. To resolve this divide between immunogenicity and tolerogenicity of IgG, we have sought to investigate the in vivo response of a single CD4+ T cell clone to an antigenic IgG mAb in various physical states. Using an adoptive transfer model, we demonstrate that aggregated and complexed Ig without adjuvant are immunogenic and elicit IgG anti-Id antibodies, while monomeric Ig induces a profound state of self-tolerance that subverts an anti-idiotypic response. This dichotomy between immunogenicity and tolerance is mirrored by differences in the early proliferation of antigen-specific CD4+ T cells and development of TFH. Finally, we show that heat-aggregated and complexed Ig, while both immunogenic, have notably different

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structural profiles and distinct requirements for CD4 T cell activation in vivo. Taken as a whole, our data suggest that immune complexes may be a common catalyst for productive activation of CD4+ T cells that drive anti-Id responses against therapeutic IgG targeting soluble antigen, in stark contrast to the tolerogenicity of these mAb in an uncomplexed deaggregated form.

Materials and Methods Mice Three background strains, A/J, C;129S4-Rag2tm1.1Flv Il2rgtm1.1Flv/J (Rag2-/-cγ-/-) and (B6 x A/ J)F1 (B6AF1) mice were bred in house. A/J CA30 Tg mice (CA30) have been described previously and were maintained on an A/J κ-/- background in-house [55, 57]. B6.SJL-PtprcaPepcb/ BoyJ (B6.SJL) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were housed in the Biological Resource Center at National Jewish Health (Denver, CO). CA30 mice were bred to B6.SJL (CA30.CD45.1) mice to create congenically marked CA30 T cells for adoptive transfers into B6AF1 mice. Mice used for experiments were generally 8–14 weeks old unless otherwise indicated and included both sexes. The National Jewish Health Institutional Animal Care and Use Committee (IACUC) approved this study and all mice were handled and bred with IACUC approval in accordance with institutional guidelines. None of the animals used in this work became ill or died prior to the experimental endpoint. If animals had exhibited symptoms of severe illness/moribundity, they would have received medical treatment or been humanely euthanized. All animals were euthanized per National Jewish Health IACUC guidelines using humane application of carbon dioxide.

Generation, purification, and storage of mAbs MAb 36–71 and mAb 36–65 were produced from ascites grown in C;129S4-Rag2tm1.1Flv Il2rgtm1.1Flv/J (Rag2-/-cγ-/-) mice that were injected with the respective hybridomas [66]. After clotting, ascites fluid was centrifuged at 20,000 x g for 30 minutes at 4˚C and passed through a 0.22 μm filter (Millipore, Billerica, MA) under sterile conditions. IgG was precipitated on ice with (NH4)2SO4 (45% v/v final), centrifuged and dissolved in phosphate buffered saline (PBS) with 0.01% NaN3. The dissolved precipitate was extensively dialyzed against 10 mM NaPO4 pH 7.9, purified by anion exchange chromatography with a DE52 resin. Residual endotoxin was removed from IgG preparations and from Ars-MSA by detergent extraction with TritonX114 according to Aida and Pabst [67]. Endotoxin levels were determined by the Limulus amebocyte lysate test [68]. Endotoxin was undetectable in all samples except those used in the TFH analysis, where it was less than 0.5 EU/sample. The DE52-purified IgG preparations were buffer exchanged from 10 mM NaPO4 pH 7.9 into a low aggregation pharmaceutical buffer (20 mM histidine, 222 mM trehalose dihydrate pH 5.5), adjusted to 4 mg/ml and passed through a 0.22 μm filter [69]. Polysorbate 80 (PS80) (Sigma Aldrich, St Louis, MO) was added to 0.02% (v/v) before freezing at -20˚C in 500 μL aliquots. Individual tubes of IgG were subjected to a single freeze-thaw cycle prior to physical analyses or injection into animals.

Generation of deaggregated, aggregated, and complexed IgG To prepare the deaggregated form, frozen samples of IgG were thawed (once only/sample), diluted in sterile PBS to a concentration of 1 mg/ml and centrifuged at 165,000 x g in a fixed angle TLA-120.1 rotor (Beckman Coulter, Brea, CA) for 3 h. The top 2/3rds of the supernatant was removed and stored at 4˚C for 30-fold). A repeat of this experiment with 5 x 104 transferred CA30 T cells gave a similar result although less robust (~8-fold reduction, S1 Fig). Recipients in this experiment were younger (6–8 weeks old) than those of the first experiment (8–14 weeks old) and likely produced more thymic emigrants during the course of the experiment, which may have encountered the complexed form of mAb 36–71 before seeing the deaggregated form. These results show that, at the level of humoral immunity, deaggregated IgG induces self-tolerance, even in the presence of excess antigen-specific T cells.

Aggregated and IC mAb 36–71 induce CA30 T cells to adopt a T follicular helper phenotype In view of the preceding results, we predicted that heat-aggregated and IC forms of mAb 36– 71 would induce the development of TFH, while the deaggregated form would not. To test this, we performed our standard adoptive transfer and immunized mice with deaggregated, heataggregated or IC forms of mAb 36–71 and then stained at day 14 for splenic TFH among CA30 T identified using the CD45.1 congenic marker (Fig 3A). While day 14 is expected to be within the contraction phase of the primary CD4+ T cell response, it is also at or near the peak of the germinal center reaction. In mice that received aggregated mAb 36–71 or IC mAb 36–71, there were noticeable percentage increases in CA30 T cells that expressed the CXCR5hiPD-1hi TFH phenotype (Fig 3B).

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Fig 1. Heat-aggregated and immune complexes of mAb 36–71 elicit an IgG anti-Id response without requiring adjuvant. (A) Adoptive transfer and immunization scheme. (B) Serum IgG anti-idiotypic antibodies directed against the Vκ of mAb 36–71 were quantified using DELFIA as described in Materials and Methods. Connected lines denote a single mouse. Data are representative of two independent experiments with n = 5 mice per group. (C) Table summarizing results from 2 experiments with n = 5 mice per group with positive titers at each time point by treatment. Both experiments had 3 mice with positive titers at d42 and 5 mice with positive titers at d63 in both the heat-aggregated and immune complex groups. doi:10.1371/journal.pone.0170556.g001

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Fig 2. Deaggregated mAb 36–71 suppresses a humoral anti-Id response against mAb 36–71. (A) Adoptive transfer and immunization scheme. Primary and secondary injections were as specified in the figure. Naïve mice were B6AF1 mice that received CA30 cells, but no primary or secondary injection. (B) Mean titration curves (above) and concentrations (below) of serum IgG anti-Id at day 21. Each point represents an experimental mouse (n = 5/group). Results representative of 2 independent experiments with n = 5 mice per group. doi:10.1371/journal.pone.0170556.g002

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Fig 3. Heat-Aggregated and IC mAb 36–71 drive development of CA30 TFH cells. (A) Adoptive transfer and immunization scheme. (B) Representative flow plots using the TFH markers PD-1 and CXCR5 or Bcl-6 and CXCR5 respectively. Cells were pregated as CD45.1+, CD4+, MHC II-, CD19-, CD8α-, F4/80-. Numbers indicate percentages of CA30 T cells with indicated TFH markers. (C) Numbers of CA30 T cells in individual mice with the indicated TFH markers. Statistical differences were determined by a two-tailed Mann-Whitney U test. Data are representative of three independent experiments with n = 5 mice per group. doi:10.1371/journal.pone.0170556.g003

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These increases were commensurate with significantly greater TFH numbers (Fig 3C), which indicated an ~8-fold increase in CA30 TFH cells in mice that received the heat-aggregated form of mAb 36–71 (p = 0.0016) and a 5.3-fold increase in those that had received the IC form (p = 0.0032) relative to the numbers in mice that received the deaggregated form. As the BCL-6 transcription repressor is critical in the development of TFH, we also confirmed increased percentages of CXCR5hiBCL6+ CA30 T cells in the groups that received heat-aggregated and IC mAb 36–71 relative to those that received the deaggregated form. In terms of CXCR5hiBCL6+ CA30 T cell numbers, the fold-increases in the heat-aggregated group and IC groups over those of the deaggregated group were nearly identical to those obtained using the PD-1hi marker.

All forms of mAb 36–71 induce efficient CA30 T cell proliferation, but aggregated and IC forms drive them through more division cycles To examine the population dynamics of early CA30 T cell proliferation, we transferred CFSElabeled CD45.1+ CA30 T cells into adoptive B6AF1 recipients and injected them with one of the 3 forms of mAb 36–71 or control mAb 36–65 the following day (Fig 4A). Five days after injection, splenocytes were assayed for CA30 T cells and CFSE profiles were examined. The congenic CD45.1+ marker allowed us to identify CA30 T cells that had divided rapidly and diluted the CFSE almost entirely. The resulting CA30 CFSE profiles revealed two notable trends (Fig 4B). First, deaggregated mAb 36–71 induced virtually all of the CA30 T cells to

Fig 4. Aggregated and IC mAb 36–71 drive CA30 T cells through more division cycles than does the deaggregated form. (A) Adoptive transfer scheme. (B) Representative FACS plots of CFSE and CD45.1 staining in the CD4+, MHC II-, CD19-, CD8α-, F4/80- gate and representative CFSE histograms for CD4+ CD45.1+ (CA30) cells. (C) Graph of percentage or total numbers (D) of CD4+ CD45.1+ (CA30) cells with SEM in each cell cycle division gate as defined by the FlowJo CFSE proliferation algorithm. Data presented are from a single experiment with mice treated with deaggregated (n = 7), immune complex (n = 7), or heat aggregated (n = 4) mAb 36–71. Statistical differences were determined by a two-tailed Mann-Whitney U test (* = p

Immunogenicity of Isogenic IgG in Aggregates and Immune Complexes.

A paradox in monoclonal antibody (mAb) therapy is that despite the well-documented tolerogenic properties of deaggregated IgG, most therapeutic IgG mA...
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